Project Summary Commonly prescribed immunosuppressant drugs target Calcineurin (CN), the ubiquitously expressed, Ca2+/calmodulin-dependent serine/threonine phosphatase, to restrict the growth and differentiation of T cells. Unfortunately, transplant patients must undergo long-term treatment with CN inhibitors, which causes unwanted side effects, including post-transplant diabetes, neurotoxicity, and cancer. These side effects are attributed to CN inhibition in non-immune cells and underscore the importance of delineating the spectrum of CN targets and functions in human cells. However, to date there are only 27 known targets of CN in humans. CN binds to substrates via Short Linear Motifs (SLiMs) termed ?PxIxIT? and ?LxVP,? which are located within intrinsically disordered regions of the proteome. PxIxIT sites in CN substrates are required for interaction with and dephosphorylation by CN and are therefore strong predictors of candidate CN substrates. We recently performed proteome-wide peptide phage display selections with CN using a library containing all known disordered regions in the human proteome, with the goal of discovering novel CN substrates and regulators. This screen, which identified PxIxIT sequences from many known CN substrates and regulators, also identified 20 novel PxIxIT-containing sequences, whose parent proteins include kinases, ion channels, cell cycle regulators, and transcription factors. These PxIxIT-containing sequences are evolutionarily conserved across all metazoans and many were independently predicted via novel computational tools developed in our laboratory. We hypothesize that many of these PxIxIT-containing proteins represent novel CN substrates and could indicate new points of regulation for CN in non-immune cells. The most highly enriched PxIxIT sequence belongs to Nup153, a nuclear basket-associated nucleoporin with a well-established role in nuclear transport. This novel PxIxIT sequence is located in a heavily phosphorylated region of Nup153 that determines interaction with nuclear transport factors, suggesting that CN may regulate the nuclear transport function of Nup153. In the first part of this proposal, we will employ in vitro and in vivo binding and dephosphorylation assays to systematically characterize these 20 PxIxIT-containing proteins and determine whether they are bona fide CN substrates. These studies will not only significantly expand the human CN signaling network, but will also provide us with new CN substrates to investigate in further mechanistic detail. In the second part of this proposal, we will investigate whether CN regulates the nuclear transport function of Nup153 by performing in vitro dephosphorylation assays and nuclear import assays in digitonin-permeabilized cells. Together, these studies will significantly expand the human CN signaling network and provide insight into novel functions of CN in non-immune cells.